Immunohistochemical identification of stent-based ablation lesions in the superior vena cava and pulmonary veins

Background: Ablation procedures, in the context of atrial fibrillation treatment, induce lesions which are difficult to visualize in an acute phase. This study sought to develop an immunohistochemical technique to evaluate and visualize microscopically the applied acute ablation lesions, which may enable an objective assessment of the lesions as this is crucial to determine lesion transmurality. Methods: In the present study, an ablation procedure was performed in vivo in six pigs at the level of the cranial vena cava and in-vitro at the level of the ostia of the pulmonary veins using the same custom-made stents and delivery system. The antibody myosin MYBPC3 was used to visualize indirectly the denaturation process induced by the heating procedure. Subsequently, the histological images were processed by using Fiji software to make standardized sections which are objectively comparable. Results: In all samples, the ablation lesions were visualized immunohistochemically and clearly identified to enable lesion measurement. The absence of transmural lesions was noticed in the samples which were exposed to an insufficient or an extreme high energy level or in the case the myocardial layer was divided by an intermediate fat layer. Conclusions: During postmortem histopathologic evaluation of ablated tissue, this technique offers a method to specifically determine the grade of muscle protein denaturation and to assess the degree of transmurality. Moreover, this study highlights the importance of myocardial tissue shielded by connective, fat and/ or charred tissue, from the penetrating ablation heat in the context of recurrence or persistence of atrial fibrillation.


Introduction
In men, atrial fibrillation (AF) is the most important cardiac arrhythmia with an increasing prevalence due to an ageing population [1,2].Besides a medical treatment of AF, ablation of myocardial sleeve tissue is increasingly used [3].AF is mainly caused by the elicitation of ectopic pulses from myocardial sleeve tissue and their conductance towards the heart [4].Ablation includes the interruption of the transfer of these pulses by using a heat or cold source.During an ablation procedure, lesions are applied to the myocardial tissue, especially at the level of the pulmonary veins wall or less commonly at the wall of the superior vena cava, coronary sinus or interatrial septum [5].During evaluation of preclinical or clinical ablation trials, a distinct histological determination of the applied lesions, in an acute stage without the presence of repair connective tissue, remains difficult as regular histological staining of ablated tissue fails to distinguish clearly denatured cells from intact cells.Consequently, an immunohistochemical technique might be doi: 10.7243/2055-091X-4-14 valuable to visualize and delineate precisely and objectively the ablated lesions and to evaluate the grade of lesion transmurality.Transmural lesions can be the cornerstone for a successful ablation procedure as the use of the appropriate energy level will define the result of creating the required local lesion [6].
However, during chronic follow-up studies based on biopsies or postmortem evaluation, ingrown connective tissue can be stained histologically to differentiate damaged from intact myocardial tissue.Different histological staining techniques for chronic lesions have been described, such as masson trichrome, hematoxylin and eosin, and elastic-van Gieson stains [6][7][8][9].
In this study, a technique is described to visualize and delineate immunohistochemically acute porcine ablation lesions at the level of the cranial vena cava (equivalent of the superior vena cava in humans) and at the level of the ostia of the pulmonary veins.

Superior vena cava
Six 1.5 month old pigs (pigs 1-6, Table 1), weighing between 40 and 45 kg were used in the animal trials performed at the IMMR institute, Paris, France according to the Ethical Committee approval number "Fulgur Medical 16-22".Pigs were anesthetized using 6 mg/kg propofol and 2 % isoflurane.In each pig, one custom-made stent was placed inside the proximal part of the cranial vena cava.More specific, by using angiography the ablation ring of the stent was positioned to target myocardial tissue.Delivery of the stent through the right auricle was performed using a custom-made delivery catheter, followed by angiographic evaluation of the stent position.

Pulmonary veins
Fresh cardiopulmonary sets of nine pigs of 60 kg (pigs 7-12, Table 1) were used in the in-vitro trials performed at the Faculty of Veterinary Medicine, Ghent, Belgium.In each set, one custom-made stent was placed inside an ostium of the pulmonary veins and afterwards the stent and the surrounding tissue was excised from the cardiopulmonary set.

Ablation procedure Superior vena cava
The stent was heated wirelessly by placing the pig in a coil to induce a magnetic field between the stent and the coil as described by Vandecasteele et al. (2016) [10].Each pig was treated with a different combination of stent diameter, ablation time and power (Table 1).

Pulmonary veins
The sample was placed inside a closed water-perfused circuit and heated wirelessly, by placing a coil around the stent and the surrounding ostium, for different time periods, flow rate and with different power settings.

Superior vena cava
All pigs were euthanized directly after the ablation proce- doi: 10.7243/2055-091X-4-14 dure.After removal of the heart-lung package, a part of the right auricle and the cranial vena cava, including the stent, were dissected.Hereafter, the stent was removed from the longitudinally opened blood vessel.The part of the vena cava demonstrating the burned marking of the stent was cut out and fixed in 4% formaldehyde solution for 24 hours.Subsequently, the samples were stored in alcohol 70%.Prior to the dehydration steps, the endothelial scar markings were stained with tissue ink (Figure 1).The dehydration procedure occurred according to standard laboratory procedures and, subsequently, the samples were embedded in paraffin using a Microm tissue processor STP 420D (Prosan, Merelbeke, Belgium) and Microm embedding station EC 350-1 (Prosan), respectively.Sections of 5 μm were cut longitudinally on a Microm H360 microtome (Prosan).

Pulmonary veins
The ablated tissue was removed from the stent and fixed, dehydrated, embedded and cut as described above.

Immunohistochemistry
The myocardial sleeve tissue of the cranial vena cava and the pulmonary veins of the different pigs was stained immunohistochemically with the polyclonal myosin marker MYBPC3 (K-16: sc-50115; Santa Cruz Biotechnology, Santa Cruz, CA, USA) to detect myosin-binding protein C (cardiac type), which is reactive with pig tissue and present in the myofibrils of cardiac and striated muscle tissue [11].Immunostaining was performed using the Dako automated Autostainer Plus.No antigen retrieval was needed.Firstly, the sections were incubated for 5 min with 3% hydrogen peroxide and 30 min with rabbit serum.After primary antibody incubation for 60 min, secondary antibody (rabbit/anti-goat, biotinylated, polyclonal, 1:500; Dako, Glostrup, Denmark) was used for 30 min followed by streptavidin and horseradish peroxidase (streptavidin-HRP, 1:1500, Dako) for 30 min.Visualization was performed with DAB (Dako) for 5 min.The immunohistochemical sections were observed under the light microscope (Olympus BX61, Olympus DP73 camera, Olympus, Belgium).Indirect lesion identification is done as follows.The antibody MYBPC3 positive regions (dark staining) will refer to intact myocardial tissue whereas the ablated parts (lighter staining) will be negative for the immunohistochemical staining.

Software processing
Free software Fiji was used to process and to standardize the images of the immunohistochemical sections by using the same threshold for each section (Figure 2) [12].After color deconvolution (for haematoxylin and DAB) and duplicating the image, the threshold was set at zero and 90.Afterwards, the adapted image was overlaid with the original image at 50% opacity and flattened together.The scale bar was set and the images were measured.

Results
The Fiji processed images showed the lesions more clearly compared to the original images as demonstrated in Figure 2.
A gradual change of brown shades was observed, depending on the position of the observed spot.If this spot is located close to the position of the applied heat source, a lighter zone is noticed compared to a spot located further away from the heat source (Figure 3).
All immunohistochemical samples of the different pigs demonstrated clear ablation lesions, except of the two control samples (pigs 14 and 15, Table 1), which are visible on the sections as lighter zones compared to the non-ablated regions.These lighter zones also correspond with the tissue ink present at the endothelial side of the ablated region of the caudal vena cava sections.Some samples displayed transmural lesions (pigs 1, 2, 3, 4, 6, 8 and 13; Figures 2-4  and 6) whereas other samples demonstrated no transmural lesions with lighter stained myosin-denaturation-patterns      at different depths, measured from the endothelium at the level of the applied ink.All samples included, the ablation depth ranged from 0.45mm to 2.38mm.
If superficially induced charring and/or physiologic connec-tive tissue was present between themyocardial tissue and the doi: 10.7243/2055-091X-4-14 heat source, little or no myosin denaturation was observed (Figure 4).Often, pulmonary vein samples demonstrated several layers of myocardial tissue separated from each other by fat and connective tissue.In that case, no myosin denaturation was noticed in the myocardial sleeve layer located at the adventitial side of the fat whereas at the same level, a transmural lesion could be observed in the myocardial tissue located at the endothelial side.Two samples of pulmonary vein tissue were ablated under the same conditions and with the same power setting.The sample in which no fat was present demonstrated an ablation lesion, more than twice the depth compared to the lesion observed in the sample in which the myocardial sleeve layer was divided in two by an intermediate layer of fat (Figure 5).
Several immunohistochemical sections of the pulmonary veins provided an ablation pattern which is in accordance with the structure of the stent.The stent is build-up by a number of struts and the diamond-like shape of the struts was recognized in the ablated tissue (Figure 6).

Discussion
The standard histological staining methods fail to make a clear distinction between destructed tissue and intact tissue.Moreover, with these techniques it is sometimes hard to visualize objectively the gradual reduction of heat intensity when sampling away from the ablation point.A misinterpretation of the grade of heat penetration, based on a regular histological staining, could be caused by an unequal fixation of the tissue, due to a variable tissue thickness as no control is available.Further, regular histological staining methods are not directly linked with the process occurring in ablated, destructed or denatured tissue but offer an overview of the present tissue types without discriminating between heated and non-heated tissue.The supra-vital staining with tetrazolium is frequently used to distinguish non-vital from vital tissue [13].However, in the case of determining the grade of transmurality, more detail or histological images on higher magnification are required which is not possible with this supra-vital staining.On the other hand, immunohistochemical staining with antibody myosin MYBPC3 visualizes exactly the gradual denaturation of the muscle protein myosin, which is, according to Tornberg (2005) [14], the first characteristic of muscle tissue that will be affected during a heating process.
The evaluation of the degree of chronic ablation lesions will offer less difficulties as ingrown connective tissue will replace destructed cardiac tissue.However, in chronic samples, the described immunohistochemical technique will also be valuable as it detects viable myocardial tissue.
The use of a too high or too low temperature during an ablation procedure will fail to achieve a transmural lesion.The application of extreme temperatures or distribution of energy on a small area will lead to superficial endothelial charring, which reduces energy transfer to the underlying cardiac muscle.On the other hand, myocardial sleeve tissue fans out distally, thus heart muscle fibers, covered by fat and connective tissue, could get shielded from the ablating heat source.Some samples, investigated in this study, demonstrated that heat penetration into the venous wall was limited due to the insulating effect of connective, fat and/or charred tissue.
This could be a reason why recurrence of ectopic electrical conductance is frequently seen after ablation procedures.The constitution of the venous wall needs to be considered in case of ablation failure, AF recurrence is noticed or electrical conductance persists.

Conclusions
This study demonstrates an immunohistochemical staining technique with the MYBPC3 antibody to visualize and to asses objectively acute ablation lesions.Moreover, the importance of the insulating role on heat transfer of endothelial charring, fat or connective tissue is emphasized.

Figure 1 .
Figure 1.Ablated superior vena cava sample of pig 1 with an applied endothelial ablation lesion, before (panel a) and after applying the blue tissue ink (panel b), scalebar=0.5cm.

Figure 2 .
Figure 2. Immunohistological image of an ablated superior vena cava of pig 1 with a transmural lesion before (left) and after (right) Fiji software processing.The ablation lesion is contoured in both images, scale bar=250μm.

Figure 3 .
Figure 3. Panel a: Fiji-software-processed immuohistological image of an ablated superior vena cava of pig 6 with tissue ink applied (insert) at the endothelial side at the level of the heating spot (between two arrows), with the latter identifiable as the myosin proteins are almost all denatured at this level, scalebar=500μm.Panel b: Detail of panel a with the insert indicating the applied ink, scalebar=100μm.Panel c: Higher magnification of the insert indicated in panel b with the arrows indicating the applied blue tissue ink, scale bar=100μm.

Figure 4 .
Figure 4. Panel a: Fiji-software-processed immunohistological image of an ablated superior vena cava of pig 4 with tissue ink applied at the endothelial side at the level of the heating spot, indicated by the arrows in panel b, scale bar of panel a=1mm.Panel b: Higher magnification of the delimited area in panel a, indicating both a non-ablated region (1) and ablated region (2), scale bar=100μm.Panel c: Highly enlarged detail of panel a, demonstrating an insulating barrier consisting of charred (1) and connective tissue (2) between the heating area (indicated by the blue ink) and the intact myocardial tissue (3), scale bar=100μm.

Figure 5 .
Figure 5.Both images (a and c) are derived from ablation trials with the same setting and conditions.Panel a: Fiji-softwareprocessed immunohistological image of an ablated pulmonary vein of pig 10.The arrow indicates an ablation lesion of approximately 2.38 mm, L =lumen, scalebar=1mm.Panel b: detail of panel a, indicated by the box, in which the loss of staining is visualized, L=lumen, scalebar= 0.5mm.Panel c: Fiji-software-processed immunohistological image of an ablated pulmonary vein of pig 11.The black arrows indicate two ablation spots (0.6mm and 1mm) in myocardial layer 1.The white arrows indicate the fat layer between myocardial layer 1 and 2, L=lumen, scalebar=1mm.Panel d: detail of panel c, indicated by the box, in which the lighter staining visualizes the transition of normal tissue to ablated tissue, L=lumen, scalebar=0.5mm.

Figure 6 .
Figure 6.Panel a: Fiji-software-processed immunohistological image of an ablated pulmonary vein of pig 12.The section was cut parallel to and 10μm from the endothelium.The arrows indicate the diamond-shaped ablation lesion which matches perfectly the organization of the struts, scale bar=2mm.Panel b: detail of panel a, indicated by the box, in which the loss of staining is indicated in higher magnification by the double arrow, scale bar=0.4mm.Panel c: Fiji-software-processed immunohistological image of an ablated pulmonary vein of pig 13.The section was cut longitudinally and the arrows indicate several transmural ablation lesions at regular distances, due to the structure of the stent.Scale bar=2mm.